The separation of complex mixtures is a task of major importance for the analyst and there are several fields now where considerable effort is made solving that kind of problems, in biochemical, clinical, environmental, food and petroleum chemistry especially.
Chromatography is by nature the most efficient separation method the analyst can use and is the backbone of all separation schemes devised. The supremacy of chromatography is a consequence of its important separation power and of the wide range of retention mechanisms which are available, most of them offering tunable selectivity by adjusting the composition of the mobile phase. It is rare, however, that the separation of a complex mixtures can be solved using one chromatographic column, either in isochratic or graident conditions.
The separation power of a chromatographic column is best expressed by its peak capacity, the number of peaks with resolution unity which are eluted between k'=0 and a final value, usually 6.4 but rarely exceeding 10. The peak capacity of a conventional packed LC column can hardly exceed a few hundreds: a hundred thousand plate column which is already a rarity, has a peak capacity between 160 (k'=6.4) and 240 (k'=20). It has been shown that assuming a Poisson distribution of the k' of the compounds of the analyzed mixture in the range of k' recorded, it cannot be used to separate much more than about 150 peaks and even for less complex mixtures a number of peaks will be multiplets, i.e. the corresponding compounds will interfere. Previous work has shown that there is little hope to increase much the peak capacity available beyond several hundreds, even by accepting very long analysis times and resorting to capillary columns of very small diameter (less than 5 micrometers) and meeting the exacting specifications their successful use demands.
Accordingly, the analysis of complex mixtures is usually attempted by combinations of selective extractions and chromatographic separations. This is time consuming, tedious, and costly because of all the additional efforts to avoid the soures of errors due to contamination and losses. So called multi-dimensional chromatography separates on a second column on fraction purified on a first column, but cannot be applied to the entire sample. An analytical scheme using several retention mechanisms integrated into a continuous series of operations applied to a sample in one single equipment would solve all these problems and simplify considerably the data handling, since all quantitative results would apply to the same sample.
Two dimension thin layer chromatography (TLC).sup.2 operates on such a principle. In this method a plate is covered with silica gel. In single phase chromatography, an unknown is placed on the plate and the plate is placed in a beaker containing enough solvent to wet the lower edge of the plate. As the solvent climbs up the silica gel layer on the plate by capillary attraction, it moves the sample along with it. However, the different constituents of the sample having different affinities for the silica gel, are separated. The development is stopped when the first constituent reaches the top of the plate. The plate is dried. The constituents can be detected as spots at different positions on the slica gel. The silica gel may also be treated in various ways: e.g., so that it is polar, in which case non-polar solvents may be utilized; or vice versa; or it may be coated with certain molecules having specific affinities for the suspected unknowns. Thus the unknowns may be separated by their polar properties, by their molecular weights or by certain chemical affinities.
The first separation using (TLC).sup.2 was published 40 years ago by Consden, Gordon and Martin. They used a 55.times.45 cm sheet of Whatman paper to separate alpha-amino acids. The first development was made with a mixture of collidine and water. It lasted 72 hours. After drying, a second development using phenol/water in an atmosphere containing coal gas and ammonia was carried out for another 48 hours. Detection was based on the ninhydrine reaction. At least 15 of the 22 proteinic amino acids were separated. Sample size was 200 micrograms of protein hydrolyzate and sensitivity 1 microgram. The spot capacity of the paper sheet can be estimated at slightly over 100.
In two-dimensional thin layer chromatography (TLC).sup.2 the chromatographic plate is coated in a strip along one edge with one form of treated or untreated silica gel and along the rest of the plate with a different form of treated or untreated silica gel. The plate is first developed along the strip by placing an edge perpendicular thereto into a beaker of appropriate solvent to separate the unknown into a linear array of constituents. After drying the plate is placed into another beaker of another solvent along the edge parallel to the linear array of constituents and they are developed up the plate to form a planar array of sub-constituents.
After drying, in order to analyze the plate, these sub-constituents must be detected and their position on the plate, and density, measured either by eye or by automatic means. Such means are expensive, and due to the spread out nature of the spots of the sub-constituents on the plate, are not very accurate.
Since there is no control of the speed of the solvent moving up the plate, reproduceability is not good. This greatly limits the efficacy of this system of analysis. In terms of quantitative analysis (derived from density measurements) results better than 10 to 20% accuracy are very difficult to obtain.
Furthermore, the development of such thin-layer chromatographic plates is time consuming, taking up to several hours in each direction. Recently however pressurized thin-layer chromatography has come into use to try to avoid this time difficulty. In this system the solvent is forced through the thin layer of a retention medium by placing a cover plate over the thin layer of retention medium. This system has increased the speed of development.
(TLC).sup.2 has developed slowly over the years. The number of publications where this technique is described or used exceeds 200, which is neither negligible nor very important. The major technological advances since the original paper have stemmed out from the progress in thin-layer chromatography (TLC) technology. The major drawbacks of the technique are (1) the difficulties in making plates with a lateral strip of a stationary phase different from the one used for the main part of the plate which makes difficult the use of retention mechanisms based on different stationary phases; (2) the cross-contamination between the two developments, i.e. the retention pattern obtained during the second development is influenced by evaporation residuals of the first solvent and is different from the retention pattern obtained directly with the second solvent on the same stationary phase; (3) the quadratic law of solvent front migration and the unfavorable relationship between the particle size and the kinetic coefficient of the plate, which prevents from using fine particles with moderately large plates and achieving large bed efficiency; (4) the sample solution is applied on the dry bed and the solvent dried out; this promotes strong selective or irreversible adsorption, clogs adsorbent particles with viscous or solid material slow to dissolve in the mobile phase, resulting in strong tailing; (5) the near impossibility to achieve directly quantitative analysis.
This last drawback is probably the most bsic and the most serious. It is already difficult to scan a conventional TLC plate. The signal does not follow the Behr law but the more complex Kubelka-Munk function. Sensitivity is moderate and the size of the scanner light beam is as large or larger than one spot standard deviation when it should be 5 times smaller. It cannot be reduced in size, however, because the noise due to the granular appearance of the surface would become too large. The compromise between sensitivity and contribution to band broadening cannot be made satisfactory. For this reason high resolution scanners such as those used for two-dimensional gel electrophoresis with a spatial resolution of 0.1 mm cannot be used with success in (TLC).sup.2 and a Vidicon Camera needs extensive digital filtering of the data, with all the difficulties associated with it.
To solve most of these problems we combine (TLC).sup.2 with over pressured development. Over pressure development was suggested by Tyihak et al for conventional thin layer chromatography (TLC). A plastic membrane is applied under pressure against the surface of the TLC plate, effectively sealing chromatographic bed between it and the plate support. The solvent can be pumped through the bed at the required velocity as long as the corresponding inlet pressure is smaller than the pressure applying the membrane. Originally invented for centrifugal TLC this technique has been extended to conventional linear TLC through the use of a distributor. This permits solving the third of the five problems discussed above.
More recently a system quite similar to those previously mentioned has been designed with the aim of achieving conditions analogous to those prevailing in high pressure liquid chromatography (HPLC) so that retention data measured by TLC can be used without correction in column chromatography. A TLC plate is protectedp by a shield which does not touch it; the end of the TLC plate protrudes out of this enclosure and is swept by a warm gas stream which vaporizes the solvent when it exits from under the shield. The plate is horizontal and the solvent phase is fed to it through a capillary siphon and a distributor. The relative positions of the plate, the tip of the siphon and the solvent tank prevents flooding the plate while supplying a sufficient amount of solvent to keep it normally wet. In these conditions a steady-state is achieved and constant speed of the mobile phase is obtained. It is not possible to adjust it, however. The speed is approximately equal to what would be derived from the quadratic law, using for plate length the distance between the distributor and the front of mobile phase resulting from its vaporization. The sample is injected with a syringe, downstream of the distributor, after the steady flow of solvent is achieved. This eliminates the difficulties associated with demixing of solvent mixtures in conventional TLC.
Excellent analytical results have been obtained with this system. Retention data compares very closely to those measured in column chromatography. Spot shape is considerably improved with drastic reduction of the trailing due to irreversible sorption by the dry adsorbent or slow dissolution of concentrated samples. A slightly modified version of this instruction has also been used to provide an elegant pilot technique for the optimization of preparative column chromatography.
The method is very simple, provides reliable results, permits considerable solvent savings and is certainly a major advance in the art of TLC, but unfortunately it has not yet received the audience it deserves. Its drawbacks are the impossibility of adjusting the solvent velocity except by changing particule size or development length, i.e. separation power, and the lack of on-line detection.
Another system is identical to the above one as far as the chromatographic part is concerned, except for the use of a wick instead of a siphon to feed the plate with the solvent. There is a signficant addition to the earlier devices, however, in the use of a UV photometer to detect the spots before they reach the exit wick. Thus a chromatogram is obtained similar to those given by an HPLC equipment, with little advantage beyond simplicity but the marked drawbacks of a flow velocity which cannot be adjusted, of a very short optical path length and a spot detection based on UV absorption carried out on a porous diffusive medium. For these reasons we do not believe that this principle is more expedient than HPLC for routine analysis.
The data published show that the resolution obtained with the new system is comparable to that achieved using the same TLC plate in a conventional manner. On the other hand the reproducibility of retention times is markedly improved, as could reasonably be expected when a method using a steady state stream, like TCC, is compared to a method using a transient-state stream like TLC, because of the complexities of the phenomena involved in TLC flow.
A more sophisticated chromatographic system has been described by Tyihak and his associates. The basic principle is to cover the sorbent layer of a TLC system by a plastic membrane applied under an external pressure. The membrane fills the irregularities of the smooth layer surface and together with the layer support makes a porous sandwich analogous to a column through which solvent can be forced under pressure. With this technique called over-pressure TLC which is indeed an actual kind of column chromatography using a column of non-conventional cros section, the velocity of the mobile phase can be adjusted at will, by setting the solvent inlet pressure at the required value, as long as it is smaller than the external pressure applying the membrane on the sorbent layer. Originally the method was limited to circular, centrifugal developement for obvious reasons of simplicity in the design, using a circular chamber with a solvent inlet at the cente,r but in a more advanced system the use of a distributor permits parallel development of a number of samples as in conventional TLC.
Experimental data demonstrate that the distance travelled by the solvent is proportional to time and not given by the quadratic law anymore, that the flow velocity of the solvent can be adjusted independently of development distance and particule size, that the retention is highly reproducible and not significantly changed by the development distance, which is not true in conventional TLC, and that the plate height does not depend anymore on the migration distance since the solvent velocity is kept constant. We note in passing that the plate height achieved, which may not be the minimum, is approximately 15 micrometers with HPTLC particles and 33 micrometers with TLC particules leading to a value of the reduced plate height of about 2-2.5, in agreement with out independent estimates of the plate performances. The solvent velocity is 0.25 mm/second in both cases, corresponding to a reduced velocity of about 1.8 in the first case and 3.5 in the second. These results are quite similar to those obtained in column chromatography. As a consequence the separation number obtained increases monotonically with solvent migration distance instead of going through a maximum smaller than 20 as it does in TLC. Needless to say, excellent analytical results are obtained.
This excellent chromatographic bed design is very close to a column with a thin rectangular profile. Its major drawback is that it is operated by starting with a dry layer, placing the samples as inconventional TLC and after pressurizing the membrane, flowing the solvent stream into the dry layer. There is no need of operating this way, although it saves solvent. Better results, especially for weakly retained compounds may be obtained using on-line injection after a steady solvent stream is established. Furthermore, this system suffers from a lack of on-line detection.
Finally, several groups have developed during these last few years a scheme to separate over a thousand different proteins and analyze them quantitatively using bi-dimensional electrophoresis. Although the separation mechanism is different from chromatography and the technological problems involved are not related, there is obviously a close relationship between the principles of the two techniques, (TCC).sup.2 and (electrophoresis).sup.2. Indeed, electrophoresis can be used for each separation mechanisms but the last in (CC).sup.2, (CC).sup.3 or (TCC).sup.2. The software used for data acquisition and handling is also quite similar.